Despite advances in the treatment of insulin dependent diabetes (IDD), a significant morbidity and mortality remains associated with long-term disease. Investigations of BB rats, NOD mice, and humans at increased risk for IDD have shown that daily prophylactic injections of insulin prior to the onset of clinical symptoms can prevent the onset of the disease. The NIH has initiated a large multi-year/multi-center Diabetes Prevention Trial (DPT-1) to test prophylactic insulin therapy for the prevention of IDD. This trial involves screening over 60,000 relatives of patients with IDD for islet cell autoantibodies (ICA). Following staging, patients will be assigned to either a high-risk (i.e., ICA positive/low first-phase insulin release [FPIR] to IVGTT; more than 50% risk for IDD) or intermediate-risk (i.e., ICA & insulin autoantibody positive/normal FPIR; 25-50% risk for IDD) categories. One-half of those meeting high-risk criteria will be given daily prophylactic subcutaneous injections of insulin and undergo an additional four days of intensive intravenous insulin therapy on an annual basis. The remaining individuals will be placed i to a non-treated control arm, but be subjected to identical follow-up analyses. Individuals at intermediate-risk for IDD will be assigned to either oral insulin or placebo therapy. The primary objective of this proposal is to identify and compare the alterations in humoral or cellular immune responsiveness to insulin and other beta cell autoantigens over time in the treated and untreated populations of individuals participating in the DPT-1. We will analyze the peripheral blood mononuclear cell responses (i.e., blastogenesis, cytokine secretion, precursor frequency) to insulin and other beta cell autoantigens (e.g., GAD 65, islet cell homogenate, etc.) in these high/intermediate-risk relatives both prior to and after prophylactic insulin therapy, as well as in the matched high/intermediate risk relatives not undergoing IDD intervention therapy. In conjunction with genetic (human leukocyte antigen DR/DQ typing) and humoral (insulin antibody, ICA, and GAD 65 autoantibody titers/subclass) immunological assessments, we aim to delineate the mechanism of disease prevention provided by insulin therapy. We hypothesize that chronic insulin administration will induce tolerization/suppression of the immune responses to islet cell antigens through bystander suppression and/or alterations in Th1/Th2 immunity to beta cell antigens. Furthermore, parenteral therapy could provide partial beta cell "rest" whereby the immunogenicity of beta cells is reduced due to a decrease in expression of autoantigens. In addition, by analyzing the untreated relatives, useful information should also be obtained regarding the natural history of events leading to the development of IDD. These findings could aid in understanding the beneficial effects for this form of immunotherapy aimed at interrupting beta cell autoimmunity and IDD.